The invention relates to methods for covalently coupling two molecular species. In particular, the invention relates to a method of coupling a hydrocarbon containing molecule to another molecule or to a support matrix. Methods of this type have many applications including enzyme-linked immunosorbant assays (ELIAS), affinity chromatography, immunocongugate preparation and the preparation of immobilized enzymes.
Heterobifunctional cross-linking agents containing two or more groups subject to independent activation allow the controlled step wise coupling of one molecular target to another.
A combined thermochemical-photochemical cross-linking agent provides the required independence of activation of the two reacting groups. In addition, the photoactivable group may be selected to allow the production, under mild reaction conditions, of a very reactive intermediate with a low level of target specificity.
It is known that certain azides, when photoactivated, produce a highly reactive nitrene intermediate capable of insertion into even carbon-hydrogen bonds.
The use of a heterobifunctional cross-linking agent having a photo-activable group with a low level of target specificity, such as an azide, is particularly advantageous in the coupling of biological molecules to a support matrix.
Examples of suitable biological molecules include various drugs, digoxin, steroids, proteins, and in fact, almost any hydrocarbon containing molecule.
In most such molecules, there will be a large number of carbon sites where coupling may occur. Although coupling at some of these sites may be precluded by steric, electrostatic or other considerations, most of these sites should be available for coupling.
Methods previously used for covalently coupling two molecules often relied on coupling at a relatively few sites on the molecules, usually at functional groups. Often such coupling would affect the molecules biological or chemical activity.
In contrast to previous methods, using the present invention, coupling at any one of the large number of carbon sites on the molecule is unlikely to have any significant effect on the molecule's biological or chemical activity. Even if coupling at a few of the carbon sites would affect the molecule's activity, there are a large number of sites available for coupling and probability considerations alone dictate that most of the coupling reactions should occur at sites where there is no significant effect on the molecule's activity.
The support matrix may be any one of a large number of natural and synthetic polymers well known for such purposes including aminopropyl and aminoaryl glass and aminohexylagarose. These and other suitable supports are available commercially in the form of beads.
The use of such heterobifunctional (thermophotochemical) cross-linking agents to covalently couple two molecular species has been reported. (See "Photochemical Immobilization of Enzymes and Other Biochemicals" by Patrick Guire in Methods in Enzymology, 44 1976, and "Photochemical Coupling of Enzymes to Mammalian Cells:, by P. Guire et al, in Pharmacological Research Communications, Volume 9, No. 1, 1977).
However, the results achieved in terms of overall coupling, particularly when coupling biological molecules as ligands to a support matrix, were quite low. This is largely thought to be the result of two problems associated with the photo-activated coupling step.
Firstly, because of the high reactivity of the nitrene intermediate coupling may occur with molecules other than the target molecule including organic solvent molecules if the reaction occurs in solution.
Secondly, the target molecule must be brought close enough to the photo-activable group for coupling to occur on photo-activation of the group.